Article Figures & Data

Figures

Effects of SeMet and METase on apoptosis and superoxide production in LNCaP cells. A, MTT assay showing a dose-dependent effect of SeMet and METase on cell viability. Cells were treated with SeMet and METase for 5 d. B, MTT assay showing a time-dependent effect of SeMet, METase, or combination on cell viability. C, flow cytometric analysis showing apoptosis (sub-G1 cell population) induced by SeMet and METase. Cells were treated with 3 μmol/L SeMet and 0.1 unit/mL METase for 24 h. D, agarose gel electrophoretic detection of DNA fragmentation as a marker of cell apoptosis induced by SeMet and METase. Cells were treated with 3 μmol/L SeMet, 0.1 unit/mL METase, and 3 μmol/L MnTMPyP alone or in combinations for 24 h. E, protection by MnTMPyP against cytotoxicity of SeMet and METase. Cells were treated with 3.0 μmol/L SeMet, 0.1 unit/mL METase, and 3.0 μmol/L MnTMPyP alone or in combinations for 5 d, and cell survival was measured by the MTT assay. F, superoxide production in cells cotreated with SeMet and METase. Cells were treated with 3.0 μmol/L SeMet and 0.1 unit/mL METase with or without 3.0 μmol/L MnTMPyP, and superoxide was measured using a chemiluminescence assay. RLU, relative light units. Points, mean of three independent experiments; bars, SD. *, P < 0.05, compared with no METase (A), SeMet or METase alone (B), and control or SeMet or METase (E and F).

Effects of p53 on cellular response to SeMet and METase in PC3 cells. A, MTT assay of dose-dependent effect of SeMet + METase on cell viability. Cells were treated with SeMet alone or with 0.1 unit/mL METase for 5 d. B, MTT assay of time-dependent effect of SeMet and METase on cell viability. Cells were treated with SeMet or METase alone or in combination. C, Western blot analysis of levels of p53, P-p53 Ser15, p21Waf1, and Bax in cells following transduction of 4 multiplicity of infection (MOI) units of empty control adenovirus (Control-Ad) or p53 cDNA adenovirus (p53-Ad) constructs for 36 h and subsequent treatment with 3 μmol/L SeMet and 0.1 unit/mL METase for 18 h. Protein loading: 40 μg for p53, P-p53 Ser15, p21Waf1, and Bax and 20 μg for β-actin. D, MTT assay of effect of p53 on viability of cells with or without SeMet and METase treatment. Cells were transduced with 4 multiplicities of infection of control-Ad or p53-Ad constructs for 36 h and then treated with 3.0 μmol/L SeMet, 0.1 unit/mL METase, or SeMet + METase for 5 d. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, compared with control-Ad with SeMet or METase only. **, P < 0.05, compared with control-Ad with SeMet + METase and p53-Ad with SeMet or METase only.

Effects of p53 on SeMet- and MET-induced production of superoxide in LNCaP and PC3 cells. A, chemiluminescence assay showing suppression of superoxide production by p53 siRNA transfection in LNCaP cells treated with SeMet and METase. B, elevation of superoxide production by cotreatment with SeMet and METase in PC3 cells transduced with Ad-p53. Cells were transfected with 50 nmol/L p53 siRNA or transduced with 4 multiplicities of infection of Ad-p53 for 36 h and then treated with 3.0 μmol/L SeMet and 0.1 unit/mL METase in suspension. Superoxide was immediately measured using a luminometer. Points, mean of three independent experiments; bars, SD. *, P < 0.05, compared with control.